Natural immunoglobulins have been known for many years, as have the various fragments thereof, such as the Fab, (Fab').sub.2 and Fc fragments, which can be derived by enzymatic cleavage. Natural immunoglobulins comprise a generally Y-shaped molecule having an antigen-binding site towards the outer end of each upper arm. The remainder of the structure, and particularly the stem of the Y, mediates the effector functions associated with immunoglobulins.
Natural immunoglobulins have been used in assay, diagnosis and, to a more limited extent, therapy. However, such uses, especially in therapy, have been hindered by the polyclonal nature of natural immunoglobulins. A significant step towards the realisation of the potential of immunoglobulins as therapeutic agents was the discovery of procedures for the production of monoclonal antibodies of defined specificity (1). However, most MAbs are produced by hybridomas which are fusions of rodent spleen cells with rodent myeloma cells. The resultant MAbs are therefore essentially rodent proteins. There are very few reports of the production of human MAbs.
Since most available MAbs are of rodent origin, they are naturally antigenic in humans and thus can give rise to an undesirable immune response termed the HAMA (Human Anti-Mouse Antibody) response. Therefore, the use of rodent MAbs as therapeutic agents in humans is inherently limited by the fact that the human subject will mount an immunological response to the MAb and will either remove it entirely or at least reduce its effectiveness.
Therefore proposals have been made for making non-human MAbs less antigenic in humans. Such techniques can be generically termed "humanisation" techniques. These techniques generally involve the use of recombinant DNA technology to manipulate DNA sequences encoding the polypeptide chains of the antibody molecule.
Early methods for humanising MAbs related to production of chimeric antibodies in which an antigen binding site comprising the complete variable domains of one antibody are fused to constant domains derived from a second antibody. Methods for carrying out such chimerisation procedures are described in EP0120694 (Celltech Limited), EP0125023 (Genentech Inc.), EP-A-0171496 (Res. Dev. Corp. Japan), EP-A-0173494 (Stanford University), and EP-A-0194276 (Celltech Limited). The Celltech EP 0194276 application discloses a process for preparing an antibody molecule having the variable domains from a mouse MAb and the constant domains from a human immunoglobulin. It also describes the production of an antibody molecule comprising the variable domains of a mouse MAb, the CH1 and CL domains of a human immunoglobulin, and a non-immunoglobulin-derived protein in place of the Fc portion of the human immunoglobulin.
Subsequently a number of further patent applications have been published relating to chimeric antibodies, including tumour specific chimeric antibodies (e.g. WO 87/02671, Int. Gen. Eng. Inc.; EP 0256654, Centocor; EP 0266663, Int. Gen. Eng. Inc. & Oncogen; WO 89/00999, Int. Gen. Eng. Inc. and EP 0332424, Hybritech Inc.). The Genentech (EP0125023) and Hybritech (EP0332424) patent applications relate to anti-carcinoembryonic antigen (anti-CEA) chimeric antibodies.
Such humanised chimeric antibodies, however, still contain a significant proportion of non-human amino acid sequence, i.e. the complete variable domains. Thus such humanised antibodies may elicit some HAMA response, particularly if administered over a prolonged period Begent et al (ref. 2)!.
In an alternative approach, described in EP-A-02394000 (Winter), the complementarity determining regions (CDRs) of a mouse MAb have been grafted onto the framework regions of the variable domains of a human immunoglobulin by site directed mutagenesis using long oligonucleotides. Such CDR-grafted humanised antibodies are less likely to give rise to a HAMA response than humanised chimeric antibodies in view of the lower proportion of non-human amino acid sequence which they contain. There are 3 CDRs (CDR1, CDR2 and CDR3) in each of the heavy and light chain variable domains.
The earliest work on CDR-grafted humanised MAbs was carried out on MAbs recognising synthetic antigens, such as the NP or NIP antigens. However, recently examples in which a mouse MAb recognising lysozyme and a rat MAb recognising an antigen on human T cells respectively were humanised have been described by Verhoeyen et al (3) and Riechmann et al (4). The preparation of the CDR-grafted antibody to the antigen on human T cells is also described in WO 89/07452 (Medical Research Council). More recently Queen et al (5) have described the preparation of a humanised CDR-grafted antibody that binds to the interleukin 2 receptor.
It has been widely suggested that immunoglobulins, and in particular MAbs, could potentially be very useful in the diagnosis and treatment of cancer (6, 7). There has therefore been much activity in trying to produce immunoglobulins or MAbs directed against tumour-specific antigens. So far, over one hundred MAbs directed against a variety of human carcinomas have been used in various aspects of tumour diagnosis or treatment (8).
There have been a number of papers published concerning the production of chimeric monoclonal antibodies recognising cell surface antigens. For instance, Sahagan et al (9) disclose a genetically engineered murine/human chimeric antibody which retains specificity for a tumour-associated antigen. Also Nishimura et al (10) disclose a recombinant murine/human chimeric monoclonal antibody specific for common acute lymphocytic leukemia antigen.
We have now prepared humanised antibodies to carcinoembryonic antigen derived from the anti-CEA mouse MAb A5B7 (11).
Our copending International Patent Application PCT/GB 90/02017 relates to the CDR-grafting of antibodies in general and describes, among other things, that antibodies having specificity for cancer markers such as CEA, e.g. the A5B7 monoclonal antibody, have been successfully CDR-grafted according to the procedure described therein.